Method and kit for detecting influenza virus, and method for diagnosing influenza virus infection

Abstract

As a technique for detecting an influenza virus with an improved accuracy, there is provided a method for detecting an influenza virus in a biological sample by using a first probe, which is decomposed by an influenza virus-derived neuraminidase and a bacterium-derived neuraminidase to generate an optically detectable signal, and a second probe, which is decomposed by the bacterium-derived neuraminidase to generate an optically detectable signal and not decomposed by the influenza virus-derived neuraminidase.

The invention claimed is: 
     
       1. A method for detecting an influenza virus in a biological sample, comprising:
 (1) step 1 of mixing the biological sample with a first probe and a second probe, wherein 
 the first probe is decomposed by an influenza virus-derived neuraminidase and a bacterium-derived neuraminidase to generate an optically detectable signal, 
 the second probe is decomposed by the bacterium-derived neuraminidase to generate an optically detectable signal and not decomposed by the influenza virus-derived neuraminidase, and 
 the signal generated from the first probe and the signal generated from the second probe can be optically discriminatorily detected, 
 (2) step 2 of detecting signals generated from the first probe and the second probe, wherein 
 if the ratio of an intensity of the signal generated from the first probe to an intensity of the signal generated from the second probe is equal to or more than a predetermined value, the presence of the influenza virus in the biological sample is detected, and 
 if the ratio is less than the predetermined value, the absence of the influenza virus in the biological sample is detected. 
 
     
     
       2. The detection method according to  claim 1 , wherein the second probe is a compound represented by the following formula (1) or a salt thereof: 
       
         
           
           
               
               
           
         
       
       wherein
 R 1 , if present, represents the same or different monovalent substituents present on a benzene ring, 
 R 2  and R 3  each independently represent a hydrogen atom, an alkyl group having to 6 carbon atoms or a halogen atom, 
 R 4  and R 5  each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom, 
 R 6  represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms or an alkyl fluoride group having to 5 carbon atoms, 
 R 7  and R 8 , if present, each independently represent an alkyl group having 1 to 6 carbon atoms or an aryl group, wherein 
 If X represents an oxygen atom, neither R 7  nor R 8  is present, 
 R 9  is, independently at each occurrence, selected from a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an alkoxy group, a hydroxy group, a carboxyl group, a halogen atom, a sulfone group, an amino group, an alkoxycarbonyl group and an oxo groups, 
 R 10 , if present, represents the same or different monovalent substituents present on a benzene ring, 
 X represents an oxygen atom, a silicon atom or a carbon atom, 
 m represents an integer of 0 to 4, 
 n represents an integer of 1 to 3, 
 s represents an integer of 1, and 
 t represents an integer of 0 to 4. 
 
     
     
       3. The detection method according to  claim 2 , wherein the first probe is 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-NANA). 
     
     
       4. The detection method according to  claim 1 , based on a digital system. 
     
     
       5. A kit for detecting an influenza virus in a biological sample isolated from a subject infected or suspected of being infected with an influenza virus, comprising:
 a first probe that is decomposed by an influenza virus-derived neuraminidase and a bacterium-derived neuraminidase to generate an optically detectable signal, and 
 a second probe that is decomposed by the bacterium-derived neuraminidase to generate an optically detectable signal and not decomposed by an influenza virus-derived neuraminidase, wherein 
 the second probe is a compound represented by the following formula (1) or a salt thereof: 
 
       
         
           
           
               
               
           
         
       
       wherein
 R 1 , if present, represents the same or different monovalent substituents present on a benzene ring, 
 R 2  and R 3  each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom, 
 R 4  and R 5  each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom, 
 R 6  represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms or an alkyl fluoride group having 1 to 5 carbon atoms, 
 R 7  and R 8 , if present, each independently represent an alkyl group having 1 to 6 carbon atoms or an aryl group, wherein if X represents an oxygen atom, neither R 7  nor R 8  is present, 
 R 9  is, independently at each occurrence, selected from a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an alkoxy group, a hydroxy group, a carboxyl group, a halogen atom, a sulfone group, an amino group, an alkoxycarbonyl group and an oxo group, 
 R 10 , if present, represents the same or different monovalent substituents present on a benzene ring, 
 X represents an oxygen atom, a silicon atom or a carbon atom, 
 m represents an integer of 0 to 4, 
 n represents an integer of 1 to 3, 
 s represents an integer of 1, and 
 t represents an integer of 0 to 4. 
 
     
     
       6. The kit according to  claim 5 , wherein the first probe is 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-NANA). 
     
     
       7. A method for diagnosing whether a subject is infected or not with an influenza virus, comprising:
 (1) step 1 of mixing a biological sample, which is isolated from a subject infected or suspected of being infected with an influenza virus, a first probe and a second probe, wherein 
 the first probe is decomposed by an influenza virus-derived neuraminidase and a bacterium-derived neuraminidase to generate an optically detectable signal, 
 the second probe is decomposed by the bacterium-derived neuraminidase to generate an optically detectable signal and not decomposed by the influenza virus-derived neuraminidase, and 
 the signal generated from the first probe and the signal generated from the second probe can be optically discriminatorily detected, 
 (2) step 2 of detecting signals generated from the first probe and the second probe, wherein 
 if the ratio of an intensity of the signal generated from the first probe to an intensity of the signal generated from the second probe is equal to or more than a predetermined value, it is determined that the subject is infected with an influenza virus, and 
 if the ratio is less than the predetermined value, it is determined that the subject is not infected with an influenza virus.